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Author Topic: antigen retrival for enzymes... will it work?  (Read 6533 times)

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Offline Dierk

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antigen retrival for enzymes... will it work?
« on: January 20, 2005, 09:59:22 AM »
HI,

I want to demonstrate tyrosinhydroxylase in rat brain sections of the substantia nigra...

Will antigen retrival using citrate buffer 10 mmol pH 6 at 5-10 min 95 C work or will my enzyme be destroyed?

Does antigen retrival generally work well with enzymes or not?

Thanks,

Dierk

antigen retrival for enzymes... will it work?
« on: January 20, 2005, 09:59:22 AM »

Offline richard03

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antigen retrival for enzymes... will it work?
« Reply #1 on: January 20, 2005, 08:43:25 PM »
Are you doing paraffin sections or free floating sections or frozen sections?

Offline Dierk

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antigen retrival for enzymes... will it work?
« Reply #2 on: January 21, 2005, 02:28:48 AM »
Hi,

I'm using rat brains which have been kept serval days at 4 C after either

1. saline perfusion + pfa perfusion + immediate post fixation at +4C using pfa immersion
or
2. saline perfusion only + immediate post fixation at +4C
using pfa immersion
or
3. no perfusion, but immediate pfa immersion at 0 C after removal of brain (freshly unfrozen pfa which is then transferred to +4C)


All brains are then cryoprotected using 20% saccharose,  and then cut on the cryostat - I'm using mostly free-floating, put I also put some directly on slide.

I'm not using any special agent (except of pfa and low temperature) which would prevent protein breakdown

Offline richard03

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antigen retrival for enzymes... will it work?
« Reply #3 on: January 21, 2005, 11:04:45 PM »
I would suggest to do perfusion using the first choice as you mentioned:

1. saline perfusion + pfa perfusion + immediate post fixation at +4C using pfa immersion

Post fixation no longer than 2 hours. Then transfer brain to 15% sucrose and then 30% sucrose in 0.1M PB, pH7.4 until brain sink to bottom of container. Freeze and cut and floating immunostaining.

There is no need to do antigen retrieval for TH staining on floating sections, but treat sections with 1% sodium borohydride in 0.1M PB for 30 minutes is recommended to improve staining intensity. After this pretreatment step, washing and blocking, then primary ab overnight...

Note: antigen retrieval with citrate buffer indeed give stronger signal comparing with none retrieval for TH staining on FFPE sections.

Richard

antigen retrival for enzymes... will it work?
« Reply #3 on: January 21, 2005, 11:04:45 PM »